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First published online 15 February 2005
doi: 10.1242/jcs.01678

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Loss of 10ß1 integrin expression leads to moderate dysfunction of growth plate chondrocytes

¹ Department of Experimental Pathology, Lund University Hospital, Box 117, 22185 Lund, Sweden
² Cartela AB, BioMedical Center I12, Sölvegatan 19, 22184 Lund, Sweden
³ Max Planck Institute of Biochemistry, Department for Molecular Medicine, Am Klopferspitz 18, 82152 Martinsried, Germany
⁴ ITI Research Institute for Dental and Skeletal Biology, University of Bern, Murtenstrasse 35, PO Box 54, CH-3010 Bern, Switzerland

View larger version (66K): [in a new window]   Fig. 1. Generation of 10 integrin-null mice. (A) Organization of the mouse 10 integrin gene, targeting construct and recombinant allele. Exons are numbered and indicated by black boxes. EGFP, enhanced green fluorescent protein; NEO, neomycin resistance gene. Relevant restriction sites: X, XbaI; N, NotI. Probe denotes the external probe used for Southern genotyping. which detects (B) Southern blot analysis of mouse tail DNA derived from progeny of heterozygous breeding (+/+, wild-type; +/–, heterozygous; –/– homozygous mutant). The 10 integrin probe indicated in A detects a 10 kb fragment and a 5.5 kb fragment in the wild type and knockout alleles, respectively. (C) RT-PCR analysis of mRNA isolated from the heart indicates a complete lack of the 10 integrin transcript in homozygous mutant (–/–) mice. st, molecular weight standards. (D) Immunohistochemical staining of the knee region from newborn wild-type and mutant mice using an 10 integrin-specific polyclonal antibody. Mutant chondrocytes show negative staining for 10 integrin. Bar, 100 µm.

View larger version (45K): [in a new window]   Fig. 2. Skeletal analysis of 10-deficient mice. (A) Staining with Alcian Blue and Alizarin Red shows no obvious skeletal defect of mutant (m) mice compared to wild-type (wt) mice at the newborn stage. (B) Skeletal staining of hindlimbs of newborn (NB), 8-week-old (8w) and 12-week-old (12w) animals. (C) X-ray analysis of 1-year-old mice indicates a slight, time progressive shortening of the long bones. The insets in C show magnified images of the wild-type and mutant femur (arrows). (D) Diagram showing the relative length of the tibia and femur of 10 integrin-null mice compared to the wild type at various ages. Bars represent mean±s.d. A significant difference in bone length was measured in mutants compared to that in control mice *P<0.05, n=5 animals per genotype.

View larger version (54K): [in a new window]   Fig. 3. Analysis of cartilage development. (A) Hematoxylin/Eosin-stained sections through the tibia of newborn mice. The epiphyseal cartilage (ec) and the length of the proliferative (p) and prehypertrophic (ph) zones are apparently normal, but the length of the hypertrophic zone (h) is reduced in the 10 integrin-deficient growth plate. In the proliferative zone, the wild type chondrocytes are flattened and show a typical stack-like arrangement. In the mutant growth plate, the proliferative chondrocytes are more rounded, and their stack-like organization is slightly impaired. (B) Electron micrographs of clusters of proliferative cells at the newborn stage. Arrows indicate degenerative cells characterized by cell shrinkage, chromatin condensation and intense cytoplasmic staining in the mutant growth plate. Note, the round shape of the mutant chondrocytes compared to the flattened shape of the control chondrocytes. (C) TUNEL assay at the newborn stage demonstrates apoptotic chondrocytes (arrows) in the proliferative (p) and hypertrophic (h) zones of the mutant growth plate but not in the control growth plate. Apoptotic chondrocytes were detectable at the perichondrium (pc) and osseo-chondrogenic junction (oc) in both the wild type and mutant. (D) Hematoxylin/Eosin-stained tibiae at 2 weeks of age. In the mutant growth plate, the columns are less organized and the chondrocytes are more rounded (compare also insets). The length of the hypertrophic zone (h) is reduced in the mutant compared to the wild type. r, resting zone. (E) Morphometric analysis demonstrating the lengths of the total growth plate (T), proliferative/prehypertrophic zone (P), hypertrophic zone (H) and the resting zone (R, at 2 weeks and 4 weeks) in wild-type (wt) and mutant (mt) mice at various ages. The lengths of the total growth plate and the hypertrophic zone were slightly, but significantly reduced in the mutant at each age group when compared to lengths at appropriate stages of the control mice (*P<0.0001). Results represent mean±s.d. of five animals per genotype and age group with three sections analyzed per animal. Bar, 100 µm (A,C,D); 50 nm (B).

View larger version (101K): [in a new window]   Fig. 4. In situ hibridization analysis for cartilage differentiation. Newborn tibial sections were hybridized with riboprobes specific for (A) collagen II (Col2a1, a marker for non-hypertrophic chondrocytes), (B) collagen X (Col10a1, a marker for hypertrophic and distal prehypertrophic chondrocytes), (C) Indian hedgehog (Ihh) and (D) PTH/PTHrP receptor (PP-R) (markers for prehypertrophic chondrocytes). In the mutant the expression domain of Col10a1 was slightly reduced compared to that in the wild type, whereas the other markers were expressed normally. ec, epiphyseal cartilage; h, hypertrophic zone; p, proliferative zone; ph, prehypertrophic zone. Bar, 100 µm.

View larger version (123K): [in a new window]   Fig. 5. Analysis of the collagen matrix in wild-type and mutant cartilage. (A,B) Electron micrographs of the pericellular and territorial (A) and the interterritorial (B) matrix compartments of wild-type and mutant growth plates at the newborn stage. Note the reduced collagen fibril density in the mutant compared to that in the wild type. (C,D) Immunohistochemical staining of the tibia for collagen type II (Col2) and type X (Col10) at the newborn stage. Collagen X was deposited in the hypertrophic (h) zone in both control and mutant mice. Note that the hypertrophic zone is smaller and that the collagen X staining extends deeper into the prehypertrophic/proliferative (p) zone in mutant cartilage. ec, epiphyseal cartilage. Bar, 400 nm (A and B); 100 µm (C and D).

View larger version (52K): [in a new window]   Fig. 6. Defective G1 progression in 10-null proliferative chondrocytes. Reduction of BrdU-labelled (A) and cyclin-D-positive (B) nuclei of mutant proliferative chondrocytes in newborn (NB) mice and in mice at 2 weeks (2w) and 4 weeks (4w) of age. Bars represent mean±s.d. *P<0.05 compared to numbers in relevant control mice. Five sections from eight to twelve animals (A) and three animals (B) were analyzed per genotype. (C) Immunohistochemistry at the newborn stage reveals elevated expression of p16 and increased nuclear translocation of Stat1 and Stat5a in the proliferative zone of mutant growth plates compared to that in the wild type (n=3 animals per genotype and n=5 sections per animal). (D) In situ hybridization shows comparable expression levels of Fgfr3 in femurs from wild-type and mutant mice at E15.5. Bar, 100 µm.

View larger version (37K): [in a new window]   Fig. 7. Assays with primary chondrocytes. (A) Normal adhesion of mutant chondrocytes to collagen type II (Col2), collagen type I (Col1), fibronectin (FN) and bovine serum albumin (BSA). The y-axis shows the adherent cells as a percentage of the total number added per well. Shown are the means±s.d. of experiments carried out in triplicate. (B) FACS analysis of integrin expression on primary chondrocytes isolated from newborn limb cartilage.

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